This invention is generally related to nitric oxide synthase inhibitors and more specifically related to treating patients having osteoarthritis with nitric oxide synthase inhibitors.
Nitric oxide (NO) is an inorganic reactive gas molecule, important in many physiological and pathological processes where it is synthesized by cells mediating vital biological functions.
Nitric oxide serves as a neurotransmitter in the brain, produced in small amounts on an intermittent basis in response to various endogenous molecular signals. Endothelial cells lining the blood vessels also produce nitric oxide in small amounts, relaxing smooth muscle and regulating blood pressure. Indeed, the production of nitric oxide has a significant effect on the function of circulating blood cells such as platelets and neutrophils as well as on smooth muscle including blood vessels and other organs. Nitric oxide is also synthesized in the immune systems. Endotoxin and cytokines induce the production of large amounts of nitric oxide in response to infectious and inflammatory stimuli, contributing to both host defense processes such as killing of bacteria and viruses as well as pathology associated with acute and chronic inflammation in a wide variety of diseases.
Nitric oxide is formed from L-arginine oxydation by at least three different isoforms of nitric oxide synthases (NOS) in mammalian cells divided into two distinct classes, constitutive and inducible. The three NOS isoforms have been identified as:
(i) Endothelial Nitric Oxide Synthase (eNOS); (Type III NOS), a constitutive, Ca++/calmodulin dependent enzyme, located in the endothelium releasing nitric oxide in response to receptor or physical stimulation;
(ii) Neuronal Nitric Oxide Synthase (nNOS); (Type I NOS), a constitutive, Ca++/calmodulin dependent enzyme, located in the brain releasing nitric oxide in response to receptor or physical stimulation; and
(iii) Inducible Nitric Oxide Sythase (iNOS); (Type II NOS), a Ca++ independent enzyme which is induced after activation of vascular smooth muscle, macrophages, endothelial cells, and a number of other cells by endotoxin and cytokines. Once expressed, this inducible NOS synthesizes large amounts of nitric oxide (NO) for long periods.
Nitric oxide generated by the constitutive enzymes acts as a transduction mechanism underlying several physiological responses. For example, eNOS is critical for production of nitric oxide, originally identified as endothelium derived relaxation factor (ERDF). The nitric oxide generated by eNOS regulates blood pressure in animals, blood flow in man, and prevents leucocyte adhesion.
On the other hand, the nitric oxide produced in large amounts by the inducible enzyme in is a cytotoxic effector molecule. As disclosed in U.S. Pat. No. 5,629,322, incorporated herein by reference, iNOS has been cloned from human liver and identified in more than a dozen other cells and tissues including smooth muscle cells, the kidney, and numerous epithelial cells in a variety of tissues including the lung and colon. This enzyme is induced upon exposure to lipopolysaccharide (LPS) and cytokines such as gamma interferon (IFN-xcex3), interleukin-1xcex2 (IL-1xcex2), and tumor necrosis factor (TNF). Once induced, nitric oxide production by iNOS continues over a prolonged period of time, and the activity of iNOS is relatively independent of intracellular Calcium concentrations.
iNOS is implicated in conditions leading to cytokine-induced hypotension including septic shock, hemodialysis and IL-2 therapy in cancer patients. The excess production of nitric oxide generated by the inducible form of nitric oxide synthase also appears to contribute to cytokine-mediated inflammation, cytotoxicity and tissue damage. accordingly, certain conditions have been identified where inhibiting nitric oxide production is advantageous. These conditions include arthritis, inflammatory bowel disease, cardivascular ischemia, diabetes, congestive heart failure, myocarditis, atherosclerosis, migraine, reflux esophagitis, diarrhea, irritable bowel syndrome, cystic fibrosis, emphysema, asthma, bronchiectasis, hyperalgesia (allodynia), cerebral ischemia (both focal ischemia, thrombotic stroke and global ischemia (secondary to cardiac arrest), multiple sclerosis and other central nervous system disorders, for example Parkinson""s disease and Alzheimer""s disease, and other disorders mediated by NO including opiate tolerance in patients needing protracted opiate analgesics, and benzodiazepine tolerance in patients taking benzodiazepines, and other addictive behavior, for example, nicotine and eating disorders.
Further conditions in which there is an advantage in inhibiting NO production from L-arginine include systemic hypotension associated with septic and/or toxic shock induced by a wide variety of agents; therapy with cytokines such as TNF, IL-1 and IL-2; as an adjuvant to short term immunosuppression in transplant therapy; and as a chemopreventative.
Although the potential uses for NOS inhibitors has been implicated in numerous diseases, the efficacy and outcome of using a NOS inhibitor to prevent, treat and cure many diseases has never been identified. For example, U.S. Pat. No. 5,629,322 at Col. 15 beginning line 60 lists an enormity of disease types where the NOS inhibitors may be used to treat a disease. However, the disease types are named as a result of speculation, without examples or analysis. Examples of other compounds which inhibit the production of nitric oxide can be found in U.S. Pat. Nos. 5,684,008 and WO 93/13055, each incorporated by reference as if written herein.
The effect and efficacy of NOS inhibitors and specifically selective iNOS inhibitors in vivo on disease progression for the many diseases has not been addressed. Therefore, the outcome and consequence of the use of the inhibitors on disease progression in vivo in many cases remains unknown. Although some information has been generated in vivo in inflammatory arthritis, which models human rheumatoid arthritis, suggesting that either non-selective or in a few cases selective NOS inhibitors reduce disease severity, there are no reports of the use of NOS inhibitors to modulate experimental models of osteoarthritis. (18, 19, 21-24)
A need exists, therefore, to determine the effect NOS inhibitors have on the progression of osteoarthritis, and targeting new uses for NOS inhibitors and methods of treatment of osteoarthritis.
Accordingly, the present invention provides new methods of treating patients with osteoarthritis by modulating: 1) the amount of synovial fluid, 2) IL-1xcex2 levels; 3) the development of osteophytes; 4) the amount of cartilage degeneration; 5) metalloprotease production; and 6) acute joint injury through administering an effective amount of an iNOS inhibitor to the patient in need thereof. Specifically, the in vivo therapeutic efficacy of a selective inhibitor of the inducible NOS, N-iminoethyl-L-lysine (L-NIL), was used to determine the effect on the progression of lesions in osteoarthritis, on joint metalloprotease production and activity, and the levels of IL1xcex2, prostaglandin E2 (PGE2) and nitrite/nitrate (the stable end products of nitric oxide) in synovial fluid.
Treatment with a selective inhibitor of iNOS reduced the severity of lesions, demonstrating the effectiveness of the inhibitor of iNOS in attenuating the progression of the disease. In addition, the inhibitor, L-NIL, reduced the production and the activity of metalloproteases in cartilage, degradative enzymes known to play a major role in the pathophysiology of osteoarthritic lesions. The effect was shown to be mediated, in part by the suppressive effect of the inhibitor on IL-1xcex2 production.
In addition, iNOS inhibitors attenuate the enhanced production of PGE2 associated with an increased production of nitric oxide at sites of inflammation. Proinflammatory action of nitric oxide and PGE2 are both suppressed by the selective iNOS inhibitor. Treatment in vivo with a selective iNOS inhibitor resulted in a marked decrease in the level of nitric oxide and PGE2 in synovial fluid, and decreased the expression of iNOS and cyclooxygenase-2 (COX-2), the enzyme repsonsible for the generation of PGE2.
This invention is the first to provide direct evidence that the in vivo suppression of nitric oxide production by selective inhibition of iNOS in osteoarthritic (OA) tissue is associated with a reduced in situ synthesis of interleukin-1 IL-1xcex2 by synovium, and metalloproteases by cartilage, as well as a reduced level of iNOS, peroxynitrite and COX-2, in OA tissue. This further elucidates the mechanisms responsible for the protective effect of the iNOS inhibitor on structural and biochemical changes seen in experimental osteoarthritis.
OA lesions in cartilage develop as a result of an imbalance in the anabolic and catabolic processes that occur during the development of the disease. (1) The excess production of nitric oxide generated by the inducible form of nitric oxide synthase contributes to cytokine-mediated inflammation, cytotoxicity and tissue damage. The present invention shows that the changes in the metabolism of chondrocytes in this disease are attributed, at least in part, to an increase in the synthesis of proinflammatory cytokines such as interluekin-1xcex2 (IFN-1xcex2). This change in the function of the chondrocytes impacts the homeostasis of the cartilage matrix. (2)
Proinflammatory cytokines accelerate degradation of the cartilage matrix. (2,3) By inducing the synthesis of proteolytic enzymes, cytokines interfere with the action of growth factors such as the insulin growth factor-1 (IGF-1) binding proteins. (4) Moreover, the inflammatory cytokines also reduce the synthesis of aggrecan, one of the major matrix macromolecules responsible for the functional properties of articular cartilage. (5,6) An increased production of nitric oxide (NO) is one of the main factors by which IL-xcex2 reduces aggrecan synthesis. (5-7).
Nitric oxide is produced in large amounts by chondrocytes upon proinflammatory cytokine stimulation. (7-13) In contrast to normal cartilage, osteoarthritic cartilage spontaneously produces nitric oxide. (12-14) High levels of stable end product of nitric oxide have been found in the synovial fluid and serum of arthritic patients. (15) Similarly, the messenger RNA and protein for inducible NO synthase, an enzyme responsible for the generation of cytotoxic levels of NO, have also been detected in synovial tissue from OA patients. (14-16).
Until the present invention, nitric oxide was only believed to contribute to the development of arthritic lesions. (17-19) This hypothesis was based on in vitro data showing that nitric oxide enhances metalloprotease (MMP) activity and inhibits proteoglycan synthesis. (8,20) Furthermore, it has been hypothesized that nitric oxide reduced the synthesis of the IL-1 receptor antagonist in chondrocytes, a process possibly responsible for the enhanced effect of IL-1 on these cells. (13).
In the practice of the subject invention, seventeen dogs were used to test the effects of a NOS inhibitor in vivo on osteoarthritis. The OA dog model was created by sectioning the anterior cruciate ligament of the right stifle joint of twelve (12) dogs by a stab wound. Dogs were divided into three (3) groups. Group I had five (5) dogs (n=5) made up of unoperated dogs that received no treatment and considered normal. Group II consists of six (6) dogs having osteoarthritis (OA dogs) without treatment. Group III consists of six (6) OA dogs that received oral L-NIL (10 mg/kg) twice daily for ten (10) weeks beginning immediately after surgery.
The knees of the dogs treated with L-NIL showed a reduction in the incidence of osteophytes compared with the untreated dogs (58% vs. 92%) as well as in their size (1.92xc2x10.58 mm vs. 5.08xc2x10.66 mm). Macroscopically, L-NIL decreased the size of the cartilage lesions both on condyles and plateaus compared with the untreated dogs by about fifty percent (50%). At the histological level, the severity of cartilage lesions on the condyles and the severity of synovial inflammation were both statistically decreased in the L-NIL treated versus the untreated dogs. Treatment with L-NIL also significantly decreased both general MMP and stromelysin activity in the cartilage and the levels of IL-1xcex2, PGE2 and nitrite/nitrate in synovial fluid.
It is the object of the present invention to provide new methods of treating osteoarthritis by administrating a therapeutic effective amount of an iNOS inhibitor to a patient in need thereof.